Autoimmune diseases result from the immune system's failure to maintain self-tolerance to antigen(s) in the affected organ. Over 40 systemic and organ-specific autoimmune diseases have been observed. Among the organ-specific autoimmune diseases are multiple sclerosis, myasthenia gravis, thyroiditis, insulin-dependent diabetes mellitus, rheumatoid arthritis and others. In spite of major and significant advances in molecular and cellular immunology in the last two decades, the molecular basis for self-tolerance and the mechanisms regulating it are still a major challenge in immunology, and autoimmune diseases remain a major medical problem. The immune-specific approaches to therapy of the disease, expected to be the most effective, have not yet yielded an effective therapy for any of the autoimmune diseases.
Accordingly, many other approaches have been investigated, some of which resulted in a limited success in decreasing the progression of the disease, such as the use of β-interferon and Copolymer 1 for treatment of multiple sclerosis, yet none of them cure the disease. Apparently, the major difficulty in devising immune specific approaches to therapy lies in the complexity of the autoimmune diseases, particularly with regard to the multiplicity of target antigens and because of the possibility that the primary target antigen(s) may be different in different patients, the difficulty in determining which of the possible target antigens is the primary target antigen for each patient, and against which of the possible epitopes on that protein the pathogenic autoimmune response is primarily directed. This is further complicated by the likely “spread of autoimmunity” as disease develops.
By way of example, multiple sclerosis (MS), an inflammatory disease of the central nervous system (CNS) characterized by neurological impairment of varying extent, results from demyelination, which is believed to result from an autoimmune response against myelin. A number of CNS myelin proteins have been postulated to be potential primary target antigens in MS on the basis of their ability to induce experimental autoimmune encephalomyelitis (EAE), a well-accepted animal model for MS, and detection of autoreactivity to these antigens in MS patients (reviewed in Kerlero de Rosbo and Ben-Nun, 1998, 1999; Kaye et al., 2000; Stevens et al., 1999; Zhong et al., 2000). Among these, myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG), the major proteins of CNS myelin, have been extensively studied (reviewed in Kerlero de Rosbo and Ben-Nun, 1998, 1999).
Activated CD4+ T cells specific for MBP or PLP are sufficient to cause EAE upon their transfer into naive syngeneic recipients, and potentially pathogenic T cells reactive against MBP or PLP have been demonstrated in MS (reviewed in Tuohy, 1994); however, comparable T cell responses to MBP or PLP were also detected in healthy individuals (reviewed in Tuohy, 1994). Thus, although specific responses to these myelin antigens are likely to be of importance in the course of the disease, they may not represent the primordial pathogenic response in MS. Consequently, in the search for antigenic specificities associated with MS, other myelin-specific, and also more recently non myelin-specific CNS antigens, have been investigated for their encephalitogenicity and/or for the presence of autoreactivity to these antigens in MS. Thus, low levels of T cell response to myelin-associated glycoprotein (MAG) and S100b, found in CNS and PNS tissues, have been observed both in MS patients and control individuals and reactivity to non nervous system-specific antigens such as heat shock proteins, transaldolase, and, to a lesser extent, 2′,3′-cyclic nucleotide 3′-phosphodiesterase, has been reported in MS (reviewed in Kerlero de Rosbo and Ben-Nun, 1998). However, none of these antigens have so far been demonstrated to be encephalitogenic, albeit T cells specific for MAG and S100b can cause CNS and PNS inflammation upon passive transfer into syngeneic mice with no clinical manifestations.
In view of the restricted localization of MS lesions to the CNS white matter, it is more likely that a primary target antigen in MS is CNS myelin-specific. Myelin proteins such as MOG, myelin-oligodendrocytic basic protein (MOBP) and oligodendrocyte-specific protein (OSP) are believed to be specific components of CNS myelin (Gardinier et al., 1992; Yamamoto et al., 1994; Bronstein et al., 1997). Our studies on the reactivity to MOG by PBL (peripheral blood lymphocytes) from patients with MS in the context of their reactivity to MBP, PLP and MAG have shown that a high proportion (50%) of MS patients react predominantly to MOG (Kerlero de Rosbo et al., 1997). Most importantly, reactivity to MOG by PBL from control individuals occurs far less frequently (Kerlero de Rosbo et al., 1997). These data, together with the demonstration of the encephalitogenic potential of MOG, strongly suggests that autoimmune reactivity to this CNS myelin-specific antigen plays an important role in the pathogenesis of MS.
Another important point emerged from our investigation of the reactivity by MS PBL to the different myelin antigens, MBP, PLP, MAG and MOG, concomitantly: 40% of the MS patients tested showed no reactivity to any of these myelin antigens. Among the several explanations which could account for this observation, one likely possibility is the involvement of autoimmune reactivity to myelin-specific antigen(s) other than MBP, PLP or MOG. In this context, we have studied the autoreactivity to MOBP, a recently uncovered CNS myelin-specific protein, which is apparently relatively abundant in CNS myelin. Our data yielded from two separate studies of the proliferative response to MOBP by PBL from MS patients and controls indicated that, out of the twenty-two patients tested overall, eleven reacted to one or several MOBP peptides whilst only four out of twenty controls tested overall reacted (Kaye et al., 2000). The demonstration by us and another laboratory that MOBP is also encephalitogenic, provides unequivocal evidence that the autoimmune reactivity observed in MS patients is potentially pathogenic and may play an important role in the pathogenesis of MS. We (Zhong et al., 2000) and another laboratory (Stevens et al., 1999) also recently demonstrated the strong encephalitogenic activity of another CNS myelin-specific protein, OSP, indicating that OSP may also be a potential target antigen for autoimmune demyelinating diseases such as MS.
A potential primary target antigen in MS could be defined as a CNS antigen which has an encephalitogenic potential, i.e. can cause EAE, and against which autoimmune reactivity can be detected in MS patients. In this context, MBP, PLP, MOG and now also MOBP can be considered potential primary target antigens, as autoreactivity against one of these antigens may play an important role in the initiation/progression of MS. In view of its high encephalitogenicity, the potential role of autoimmune responses to OSP in the pathogenesis of MS should also be considered. In contrast, autoimmune responses to other nonencephalitogenic CNS components, myelin-specific or non myelin-specific, which can be detected in MS, are more likely to represent secondary events resulting from “autoimmune spread” as a result of inflammation within CNS with ongoing disease. The multiplicity of potential primary target antigens in MS points to the complexity of the disease with regard to possible pathogenic processes involved, possible etiology of the disease, and most importantly, it imposes major difficulties in devising immune-specific therapeutic approaches to MS.
Thus, the major problems that must be addressed by immune-specific therapies for a given autoimmune disease include the multiplicity of potential primary target antigens with the possibility that the primary target antigens differ in different patients, and the recently acknowledged “spreading of autoimmunity” as disease develops. This phenomenon is described as the observation of variation in the active immunogenic epitopes with the progression of the disease. This results in the evolution of the primary T cell response focused on a particular self-antigen, towards the recruitment of T cells to multiple antigenic determinants on this or other potential target autoantigens within the affected organ (Tuohy et al., 1998; Kumar, 1998).
PCT International Publication WO 01/31037 of the present applicants discloses synthetic human target autoantigen genes comprising sequences coding for at least two immunogenic epitope clusters (hereinafter EEC) of autoantigen(s) related to a specific autoimmune disease, wherein said at least two IECs may be derived from a sole autoantigen or from at least two different autoantigens related to said autoimmune disease, and polypeptides encoded thereby, that can be used for the treatment and diagnosis of autoimmune diseases such as multiple sclerosis (MS), insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA) and others. Several synthetic human genes have been disclosed in said WO 01/31037, each gene comprising sequences coding for at least two IECs of autoantigen(s) related to a specific autoimmune disease such as MS, IDDM or RA, said synthetic gene being selected from:(i) a synthetic human target autoantigen gene (designated shTAG) comprising nucleotide sequences coding for at least two IECs of a sole autoantigen related to said autoimmune disease; and (ii) a synthetic human multitarget autoantigen gene (designated shMultiTAG) comprising nucleotide sequences coding for at least one IEC of at least two different autoantigens related to said autoimmune disease. WO 01/31037 further disclosed several synthetic polypeptides, each polypeptide comprising amino acid sequences of at least two IECs of autoantigens related to a specific autoimmune disease such as MS, IDDM or RA, said synthetic polypeptide being selected from: (i) a synthetic human polypeptide (designated shPEP) comprising amino acid sequences of at least two IECs of a sole autoantigen related to said autoimmune disease; and (ii) a synthetic human multitarget polypeptide (designated shMultiPEP) comprising amino acid sequences of at least one IEC of at least two different autoantigens related to said autoimmune disease.
In MS, the multiplicity of potentially pathogenic autoreactivities against the myelin components, MBP, PLP, MOG, MOBP, and OSP, which have been detected in different patients, suggest that the primary target antigen(s) and/or the major epitope(s) against which the dominant pathogenic autoreactivities are directed, may differ in different patients. Because neoreactivities are also likely to emerge, disease progression may be associated with multiple potentially pathogenic T cell autoreactivities. This imposes major difficulties for devising immune-specific approaches for therapy of MS.
Thus, effective immune-specific therapy would have to be tailored for each patient according to the antigenic and epitope specificities of the potentially pathogenic autoimmune T-cells detected in that patient, including novel T-cell specificities elicited as a result of “autoimmune spread” as disease progresses. An alternative and more generally applicable approach would ideally be, if all or most of relevant potentially pathogenic autoreactivities could be targeted concomitantly. This would allow immunomodulation of MS, irrespective of the antigenic primacy or dominance of the pathogenic autoimmune response in individual patients.
Several studies in EAE strongly suggest that neutralizing T cells specific for one epitope may not be a sufficiently effective therapeutic approach for disease associated with multiple pathogenic autoreactivities. Thus, in chronic EAE induced in SJL/J mice with whole PLP, tolerization with the major encephalitogenic peptide, PLP139-151, abrogated the primary acute phase, but not subsequent relapses related to autoimmune spread. The clinical severity of EAE induced in (PL/J×SJL/J) F1 mice with a combination of MBP and MOG could be significantly reduced by tolerogenic administration of a combination of the immunodominant encephalitogenic epitopes within MBP Ac1-11 and MOG41-60 (Leadbetter et al., 1998). In contrast, MBPAc1-11, which suppresses MBP-induced EAE in these F1 mice, had no effect on MOG-induced EAE and a marginal therapeutic effect on EAE induced by the MBP/MOG combination (Leadbetter et al., 1998), an observation most likely related to its specific suppressive effect on MBP-reactive T cells. Highly relevant to treatment of disease with multiple autoreactivities is the strong therapeutic effect on EAE of MP4, a chimeric fusion protein, comprising the whole long isoform of MBP (21.5 kDa MBP) and the hydrophilic domains of PLP (ΔPLP) (Elliott et al., 1996).
Tolerogenic administration of MP4 fully abrogated EAE actively induced with PLP139-151, as well as EAE adoptively transferred with a combination of encephalitogenic MBP- and PLP-specific T cells in SJL/J mice. In contrast, neither 21.5 kDa MBP nor ΔPLP injected individually had any such dramatic effect on passive EAE mediated by the combined T cell populations (Elliott et al., 1996). Taken together, these studies suggest that targeting the majority of relevant T cells may be required for optimal efficacy of immune-specific therapy in disease associated with pathogenic T cell reactivities against more than one antigen/epitope.
In summary, the state of the art has convincingly demonstrated that, while EAE resulting from autoreactivity to a single autoantigen can be effectively suppressed by neutralization of the relevant T-cells via tolerogenic administration of the relevant epitope, a single epitope is not effective in suppressing EAE associated with multiple pathogenic autoreactivities. In contrast, targeting all relevant T-cells concomitantly can result in full abrogation of disease.